Fusion between two eukaryotic cells is a fundamentally interesting and biologically important process. We have only a rudimentary understanding of how cell fusion occurs. This grant proposal focuses on characterizing the process of anastomosis (vegetative hyphal cell fusion) in the filamentous fungus Neurospora crassa. The major goals of the proposed research are to identify and characterize proteins that function to mediate the cell fusion event and to determine how they function to facilitate cell fusion. The genes that are required for cell fusion, as defined by cell fusion-defective mutants, will be identified and characterized. The mutations responsible for the cell fusion-defective phenotype will be mapped to small regions of the Neurospora genetic map by classical genetic mapping techniques. The affected genes will be identified using a PCR amplification and DNA sequencing strategy. The use of this positional cloning approach has been made possible by the publication of the Neurospora genomic DNA sequence. The identity of the genes encoding cell fusion proteins will be verified by gene disruption experiments. Anastomosis can be characterized as a series of steps leading to a cell fusion event. To help us determine which proteins function is each of these steps, differential interference contrast microscope and confocal microscope time-lapse video recording systems will be used to characterize the cell fusion mutants and elucidate which cell fusion steps are blocked within these mutants. We will also determine the location and follow the movement of a few key cell fusion proteins during cell fusion events. The cellular location of cell fusion proteins will be determined in immunolocalization experiments. Key cell fusion proteins will be tagged with the green fluorescent protein and the movement of these tagged proteins during cell fusion events will be followed with the confocal microscope time-lapse video recording system. We anticipate that these experiments will provide us with important insights into how cell fusion events are orchestrated. Public Health Relevance: Cell fusion events are critical to the process of fertilization and for the differentiation of muscle, bone, and placenta. The proposed research will help us better understand cell fusion events. [unreadable] [unreadable] [unreadable]